Antithrombin (AT) is a plasma serine protease inhibitor (serpin) that regulates the proteolytic activity of coagulation proteases in plasma. AT, however, is a weak inhibitor unless it is activated by heparin-like glycosaminoglycans that line the microvasculature. The structure of AT is consisted of three beta-sheets (A to C) and nine alpha-helices (A to I). Structural data suggests that the low inhibitory activity of AT is due to insertion of two N-terminal P14 and P15 residues of the reactive site loop into the central beta-sheet A of the molecule. This leads to hiding of P1-Arg of the inhibitor. P1-Arg functions as a bait to trap coagulation enzymes in inactive states. AT is activated when a distinct pentasaccharide sequence of heparin binds to helix D of the serpin. This induces a conformational change in the inhibitor that leads to both expulsion of N-terminal residues, and exposure of P1-Arg of the inhibitor. The activation process is also linked to exposure of a new exosite on AT that enables the inhibitor to interact with an unknown exosite on coagulation proteases. For unknown reasons, however, the heparin activation of AT improves the inhibitory activity of AT with some proteases like factors Xa and IXa, but not others like thrombin. In the case of thrombin, high molecular weight heparins accelerate the inhibition reaction by an alternative template mechanism. In this application, we propose to prepare several AT and coagulation protease mutants to investigate 1) the mechanism by which heparin activation of AT enhances the reactivity of the serpin with coagulation proteases; 2) the mechanism by which coagulation proteases discriminate between the native and activated conformations of AT; 3) the effectiveness of therapeutic heparins in mediating the AT inhibition of factor Xa when the protease is assembled into the prothrombinase complex to convert prothrombin to thrombin; 4) the structural basis for the loop insertion that prevents AT from adopting an inhibitory conformation; and 5) the mechanism by which complex formation of thrombin with the endothelial cell cofactor, thrombomodulin, renders the protease susceptible to rapid inhibition by another serpin, protein C inhibitor, without changing its reactivity with AT.